Project Summary Transposable elements (TEs) are discrete mobile DNA sequences whose intact and mutated copies constitute a substantial fraction of host genomes. Over the past decade, numerous studies implicated TEs in the evolution of genomes and gene expression networks. However, whether TEs contribute to development or disease is an open crucial question answering which might expand and even radically change our views on the genetic foundations of human health and pathology. Of particular interest to human health is a Long Interspersed Element 1 (LINE-1 or L1). For example, over a hundred single-gene human diseases arose from L1 insertions. Intriguingly, recent studies implicated L1 retrotransposition in normal development (embryogenesis, oogenesis, neurogenesis), aging and disease (autoimmune disorders, cancers). However, despite a multitude of correlative studies, none have established a causative role of L1 retrotransposition in these processes to date. The primary challenge of studying endogenous L1s lies in the large numbers of full-length intact L1s in mammalian genomes. Experimental reduction of TE activity at the transcriptional or post-transcriptional levels does not provide specific, stable and long-term L1 repression in complex tissues. This R21 proposal aims to make the first step toward closing this gap in our understanding of the contribution of L1 to development and disease. We propose an experimental strategy to gradually and systematically inactivate full-length L1 elements in the mouse genome using the embryonic stem cell (ESC) model. We will monitor and quantify the extent of L1 mutagenesis and ultimately identify candidate ESC clones whose developmental potential we will subsequently test in vivo. The proposed research will provide the foundation for future rigorous tests for the role of L1s in development and disease. Relevance Studies over the past decade implicated mobile element retrotransposon L1 in healthy development and disease. If true, this contention will radically expand our understanding of the genetic foundations of human health and pathology. We propose to test this hypothesis by systematically inactivating L1 elements in the genome of mouse embryonic stem cells and characterizing the developmental potential of genomes lacking active L1 elements.